Vascular endothelial growth factor (VEGF) consists of at least six isoforms with various numbers of amino acids (121, 145, 165, 183, 189, and 206 amino acids) produced through alternative splicing (1). VEGF121, VEGF165, and VEGF189 are the forms secreted by most cell types and are active as homodimers linked by disulfide bonds. VEGF121 does not bind to heparin like the other VEGF species (2). VEGF is a potent angiogenic factor that induces proliferation, sprouting, migration, and tube formation of endothelial cells. There are three high-affinity tyrosine kinase VEGF receptors on endothelial cells (VEGFR-1, Flt-1; VEGFR-2, KDR/Flt-1; and VEGFR-3, Flt-4). Several types of non-endothelial cells such as hematopoietic stem cells, melanoma cells, monocytes, osteoblasts, and pancreatic β cells also express VEGF receptors (1).
VEGF receptors were found to be overexpressed in various tumor cells and tumor-associated endothelial cells (3). Inhibition of VEGF receptor function has been shown to inhibit pathological angiogenesis as well as tumor growth and metastasis (4, 5). Radiolabeled VEGF has been developed as a tracer for imaging solid tumors and angiogenesis in humans (6-8). Alanine scanning mutagenesis in the corresponding domains in VEGF revealed that charged amino acid residues in two exposed domains of VEGF are involved in binding to VEGFR-1 (Asp63, Glu64, and Glu67) and VEGFR-2 (Arg82, Lys84, and His86), respectively. Wang et al. (9) prepared a D63AE64AE67A mutant of VEGF121 (VEGFDEE) by changing Asp63, Glu64, and Glu67 to Ala63, Ala64, and Ala 67 so that the mutant could bind preferably to VEGFR-2 over VEGFR-1. VEGFDEE was labeled with 64Cu via 1,4,7,10-tetraazacyclododecane-N,N',N'',N'''-tetraacetic acid (DOTA) for imaging VEGFR-2 expression in an orthotopic 4T1 murine breast tumor model to develop 64Cu-labeled DOTA-VEGFDEE (64Cu-DOTA-VEGFDEE) as a single-photon emission computed tomography (PET) imaging agent to study tumor angiogenesis.